Search Results (17)

Drought is one of the major abiotic stresses that inhibits plant growth and development. Therefore, it is critical to explore drought resistance genes in crops to obtain high-quality breeding materials. In this study, the drought-sensitive tomato line “FQ118” and the resistant line “FQ119” were treated with PEG-6000 and, at 0 h (CK), 6 h, 24 h, 36 h and 48 h, the plants were evaluated for growth and physiological indicators, and leaf tissues were collected for RNA-seq. The growth indicators (growth trend, dry and fresh weights above- and below-ground, etc.) and the antioxidant enzyme system reflect that “FQ119” has stronger drought tolerance. Through RNA-seq analysis, a total of 68,316 transcripts (37,908 genes) were obtained. The largest number of significant differentially expressed genes (DEGs) in the comparison of “FQ118” and “FQ119” was observed at 6 h and 48 h. KEGG analysis demonstrated the significant enrichment of certain pathways associated with drought stress, such as glycerolipid metabolism and galactose metabolism. Co-expression analysis revealed that 7 hub DEGs, including genes encoding a photosystem reaction center subunit protein, chlorophyll a-b binding protein, glyceraldehyde-3-phosphate dehydrogenase A (GAPDH), and others, were coenriched in both comparisons. In addition, three hub genes specific to the comparison during the 6-h processing stage, encoding oxygen-evolving enhancer protein 1, receptor-like serine/threonine-protein kinase and calcium-transporting ATPase, were identified. The above hub genes were related to plant resistance to drought stress, and RT‒qPCR verified that the overall magnitudes of the differences in expression between the two lines gradually increased over time. Virus-induced gene silencing (VIGS) experiments have demonstrated that GAPDH plays a relevant role in the drought resistance pathway. In addition, the differences in expression of 7 DEGs encoding transcription factors, including Dofs, WRKYs, MYBs, and MYCs, also tended to increase with increasing duration of drought treatment, as determined via qPCR. In summary, this study identified several valuable genes related to plant drought resistance by screening genes with differential transcription under drought stress. This in-depth gene mining may provide valuable references and resources for future breeding for drought resistance in tomato. Full article

Antimicrobial-resistant (AMR) bacteria pose a significant global health threat, and bacteria that produce New Delhi metallo-β-lactamase (NDM) are particularly concerning due to their resistance to most β-lactam antibiotics, including carbapenems. The emergence and spread of NDM-producing genes in food-producing animals highlight the need for a fast and accurate method for detecting AMR bacteria. We therefore propose a PCR-coupled CRISPR/Cas12a-based fluorescence assay that can detect NDM-producing genes (bla_NDM) in bacteria. Thanks to its designed gRNA, this CRISPR/Cas12a system was able to simultaneously cleave PCR amplicons and ssDNA-FQ reporters, generating fluorescence signals. Our method was found to be highly specific when tested against other foodborne pathogens that do not carry bla_NDM and also demonstrated an excellent capability to distinguish single-nucleotide polymorphism. In the case of bla_NDM-₁ carrying E. coli, the assay performed exceptionally well, with a detection limit of 2.7 × 10⁰ CFU/mL: 100 times better than conventional PCR with gel electrophoresis. Moreover, the developed assay detected AMR bacteria in food samples and exhibited enhanced performance compared to previously published real-time PCR assays. Thus, this novel PCR-coupled CRISPR/Cas12a-based fluorescence assay has considerable potential to improve current approaches to AMR gene detection and thereby contribute to mitigating the global threat of AMR. Full article

Salmonella enterica serovar Indiana (S. Indiana) is among the most prevalent serovars of Salmonella and is closely associated with foodborne diseases worldwide. In this study, we combined a recombinase polymerase amplification (RPA) technique with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) protein Cas12b (CRISPR/Cas12b)-based biosensing in a one-pot platform to develop a novel one-step identification method for S. Indiana infection diagnosis. The entire RPA-CRISPR/Cas12b reaction can be completed at 41 °C within 1 h without the need for specific instruments. The optimal concentrations of Cas12b and single-guide RNA (sgRNA) for the reaction were the same at 250 nM. The single-stranded DNA (ssDNA) reporter 8C-FQ (5′-/6-FAM/CCCCCCCC/BHQ1/-3′) presented the best performance in the reaction compared with the other reporters. The limit of detection (LoD) of the RPA-CRISPR/Cas12b assay was 14.4 copies per reaction. As for specificity, we successfully identified four S. Indiana strains among twenty-two Salmonella strains without any false-positive results, presenting 100% accuracy for S. Indiana, and no cross-reactions were observed in eight other pathogens. Moreover, a total of 109 chicken carcasses were classified by the S. Indiana RPA-CRISPR assay and PCR methods from three processing points, including 43 post-shedding, 35 post-evisceration, and 31 post-chilling. There were 17 S. Indiana-positive samples identified during the whole processing step, consisting of nine post-shedding, five post-evisceration, and three post-chilling. The corresponding S. Indiana-positive rates of post-shedding, post-evisceration, and post-chilling were 20.93% (9/43), 14.29% (5/35), and 9.68% (3/31), respectively. Results from the S. Indiana one-step RPA-CRISPR/Cas12b assay were totally in agreement with those obtained using a traditional culture method, demonstrating 100% agreement with no false-positive or false-negative results observed. Altogether, the RPA-CRISPR/Cas12b assay developed in this study represents a promising, accurate, and simple diagnostic tool for S. Indiana detection. Full article

Traumatic brain injury (TBI) is a major cause of mortality and disability around the world, for which no treatment has been found. Nociceptin/Orphanin FQ (N/OFQ) and the nociceptin opioid peptide (NOP) receptor are rapidly increased in response to fluid percussion, stab injury, and controlled cortical impact (CCI) TBI. TBI-induced upregulation of N/OFQ contributes to cerebrovascular impairment, increased excitotoxicity, and neurobehavioral deficits. Our objective was to identify changes in N/OFQ and NOP receptor peptide, protein, and mRNA relative to the expression of injury markers and extracellular regulated kinase (ERK) 24 h following mild (mTBI) and moderate TBI (ModTBI) in wildtype (WT) and NOP receptor-knockout (KO) rats. N/OFQ was quantified by radioimmunoassay, mRNA expression was assessed using real-time PCR and protein levels were determined by immunoblot analysis. This study revealed increased N/OFQ mRNA and peptide levels in the CSF and ipsilateral tissue of WT, but not KO, rats 24 h post-TBI; NOP receptor mRNA increased after ModTBI. Cofilin-1 activation increased in the brain tissue of WT but not KO rats, ERK activation increased in all rats following ModTBI; no changes in injury marker levels were noted in brain tissue at this time. In conclusion, this study elucidates transcriptional and translational changes in the N/OFQ-NOP receptor system relative to TBI-induced neurological deficits and initiation of signaling cascades that support the investigation of the NOP receptor as a therapeutic target for TBI. Full article

The aac(6′)-Ib gene is the most widespread gene encoding aminoglycoside-modifying enzyme and conferring resistance to tobramycin, streptomycin and kanamycin. The variant aac(6′)-Ib-cr gene confers resistance to both aminoglycosides and fluoroquinolones (FQ). A total of 132 Campylobacter isolates, including 91 C. jejuni and 41 C. coli, were selected from broiler hens isolates. The aac(6′)-Ib gene was amplified using PCR and was subsequently digested with the BtsCI restriction enzyme to identify aac(6′)-Ib-cr. Among these isolates, 31 out of 41 C. coli (75.6%) and 1 (0.98%) C. jejuni were positive for the aac(6′)-Ib gene, which was identified as the aac(6′)-Ib-cr variant in 10 (32.25%) C. coli isolates. This variant was correlated with mutations in gyrA (Thr-86-Ile), as well as resistance to FQs. This study is the first report in Tunisia on Campylobacter coli strains harboring both the aac(6′)-Ib and aac(6′)-Ib-cr variants. These genes were present in Campylobacter isolates exhibiting resistance to multiple antibiotics, which restricts the range of available treatments. Full article

Sclerotinia ginseng is the primary pathogenic fungus responsible for Sclerotinia rot of ginseng, which significantly reduces plant yield and quality. The density of sclerotia in the soil is closely associated with rot incidence and severity. Whole genome sequencing was conducted to obtain fungal frame maps. The specific primers, q2001F/q2001R, were screened out by pan-genomic analysis using the NCBI database. Recombinant plasmids containing amplicons obtained with this primer set were used as standard plasmids to construct a real-time fluorescence quantitative PCR system. The relationships between the cycle threshold (Ct) values and the soil sclerotium densities were determined by real-time PCR. Real-time PCR had a detection limit of 1.5 × 10⁻² g·kg⁻¹ soil for Sclerotinia rot causing fungal mycelium, and the relationship between the density of S. ginseng mycelium n (g·g⁻¹ soil) and the Ct value was n = 10^{(40.048 − Ct)/6.9541}. The detection limit of real-time PCR for measuring soil sclerotia was 3.8 × 10⁻⁵ g·g⁻¹ soil, suggesting a sensitivity 100 times that of conventional PCR. The relationship between the sclerotium density n (g·g⁻¹ soil) and the Ct value was n = 10^{(18.351 − Ct)/7.0914}. Compared with the conventional PCR method, the fluorescent quantitative PCR method could detect the population of Sclerotinia spp. in soil more efficiently, accurately, and sensitively. Full article

Fluoroquinolones (FQs) are recommended as prophylaxis for men undergoing transrectal prostate biopsy (TRUS-Bx). Recent studies suggest a significant share of FQ-resistant rectal flora in post-TRUST-Bx infections. Methods: 435 Enterobacterales isolates from 621 patients attending 12 urological departments in Poland were screened by PCR for PMQR genes. PMQR-positive isolates were tested for quinolone susceptibility and investigated by whole genome sequencing (WGS) methods. Results: In total, 32 (7.35%) E. coli strains with ciprofloxacin MIC in the range 0.125–32 mg/L harbored at least one PMQR gene. qnrS and qnrB were the most frequent genes detected in 16 and 12 isolates, respectively. WGS was performed for 28 of 32 PMQR-producing strains. A variety of serotypes and sequence types (STs) of E. coli was noticed. All strains carried at least one virulence gene. AMR genes that encoded resistance against different classes of antibiotics were identified. Additionally, five of 13 ciprofloxacin-susceptible E. coli had alterations in codon 83 of the GyrA subunits. Conclusion: This study provides information on the common presence of PMQRs among E. coli, which may explain the cause for development of post-TRUS-Bx infections. High numbers of virulence and antimicrobial resistance genes detected show a potential for analysed strains to develop infections. Full article

The evolution of resistance in Salmonella to fluoroquinolones (FQs) under a broad range of sub-inhibitory concentrations (sub-MICs) has not been systematically studied. This study investigated the mechanism of resistance development in Salmonella enterica serovar Enteritidis (S. Enteritidis) under sub-MICs of 1/128×MIC to 1/2×MIC of enrofloxacin (ENR), a widely used veterinary FQ. It was shown that the resistance rate and resistance level of S. Enteritidis varied with the increase in ENR concentration and duration of selection. qRT-PCR results demonstrated that the expression of outer membrane porin (OMP) genes, ompC, ompD and ompF, were down-regulated first to rapidly adapt and develop the resistance of 4×MIC, and as the resistance level increased (≥8×MIC), the up-regulated expression of efflux pump genes, acrB, emrB amd mdfA, along with mutations in quinolone resistance-determining region (QRDR) gradually played a decisive role. Cytohubba analysis based on transcriptomic profiles demonstrated that purB, purC, purD, purF, purH, purK, purL, purM, purN and purT were the hub genes for the FQs resistance. The ‘de novo’ IMP biosynthetic process, purine ribonucleoside monophosphate biosynthetic process and purine ribonucleotide biosynthetic process were the top three biological processes screened by MCODE. This study first described the dynamics of FQ resistance evolution in Salmonella under a long-term selection of sub-MICs of ENR in vitro. In addition, this work offers greater insight into the transcriptome changes of S. Enteritidis under the selection of ENR and provides a framework for FQs resistance of Salmonella for further studies. Full article

In recent times, resistant foodborne pathogens, especially of the Campylobacter species, have created several global crises. These crises have been compounded due to the evolution of multidrug-resistant (MDR) bacterial pathogens and the emergence of extensively drug-resistant (XDR) and pan-drug-resistant (PDR) strains. Therefore, this study aimed to investigate the development of resistance and the existence of both XDR and PDR among Campylobacter isolates. Moreover, we explored the use of the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) technique for the detection of fluoroquinolone (FQ)-resistant Campylobacter isolates. A total of 120 Campylobacter isolates were identified depending on both phenotypic and genotypic methods. Of note, cefoxitin and imipenem were the most effective drugs against the investigated Campylobacter isolates. Interestingly, the majority of our isolates (75%) were MDR. Unfortunately, both XDR and PDR isolates were detected in our study with prevalence rates of 20.8% and 4.2%, respectively. All FQ-resistant isolates with ciprofloxacin minimum inhibitory concentrations ≥4 µg/mL were confirmed by the genetic detection of gyrA chromosomal mutation via substitution of threonine at position 86 to isoleucine (Thr-86-to-Ile) using the PCR-RFLP technique. Herein, PCR-RFLP was a more practical and less expensive method used for the detection of FQ resistant isolates. In conclusion, we introduced a fast genetic method for the identification of FQ-resistant isolates to avoid treatment failure through the proper description of antimicrobials. Full article

Background: medication-assisted treatment (MAT) with buprenorphine is now widely prescribed to treat addiction to heroin and other illicit opioids. There is some evidence that illicit opioids enhance HIV-1 replication and accelerate AIDS pathogenesis, but the effect of buprenorphine is unknown. Methods: we obtained peripheral blood mononuclear cells (PBMCs) from healthy volunteers and cultured them in the presence of morphine, buprenorphine, or methadone. We infected the cells with a replication-competent CCR5-tropic HIV-1 reporter virus encoding a secreted nanoluciferase gene, and measured infection by luciferase activity in the supernatants over time. We also surveyed opioid receptor expression in PBMC, genital epithelial cells and other leukocytes by qPCR and western blotting. Reactivation from latency was assessed in J-Lat 11.1 and U1 cell lines. Results: we did not detect expression of classical opioid receptors in leukocytes, but did find nociception/orphanin FQ receptor (NOP) expression in blood and vaginal lymphocytes as well as genital epithelial cells. In PBMCs, we found that at physiological doses, morphine, and methadone had a variable or no effect on HIV infection, but buprenorphine treatment significantly increased HIV-1 infectivity (median: 8.797-fold increase with 20 nM buprenorphine, eight experiments, range: 3.570–691.9, p = 0.0078). Using latently infected cell lines, we did not detect reactivation of latent HIV following treatment with any of the opioid drugs. Conclusions: our results suggest that buprenorphine, in contrast to morphine or methadone, increases the in vitro susceptibility of leukocytes to HIV-1 infection but has no effect on in vitro HIV reactivation. These findings contribute to our understanding how opioids, including those used for MAT, affect HIV infection and reactivation, and can help to inform the choice of MAT for people living with HIV or who are at risk of HIV infection. Full article

Thermophilic Campylobacter species of poultry origin have been associated with up to 80% of human campylobacteriosis cases. Layer chickens have received less attention as possible reservoirs of Campylobacter species. Initially, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of two archived Campylobacter isolates (Campylobacter jejuni strain 200605 and Campylobacter coli strain 200606) from layer chickens to five antimicrobials (ciprofloxacin, nalidixic acid, erythromycin, tetracycline, and gentamicin) were determined using broth microdilution while the presence of selected antimicrobial resistance genes was performed by polymerase chain reaction (PCR) using specific primers. Whole-genome sequencing (WGS) was performed by the Illumina HiSeq X platform. The analysis involved antimicrobial resistance genes, virulome, multilocus sequence typing (MLST), and phylogeny. Both isolates were phenotypically resistant to ciprofloxacin (MIC: 32 vs. 32 µg/mL), nalidixic acid (MIC: 128 vs. 64 µg/mL), and tetracycline (MIC: 64 vs. 64 µg/mL), but sensitive to erythromycin (MIC: 1 vs. 2 µg/mL) and gentamicin (MIC: 0.25 vs. 1 µg/mL) for C. jejuni strain 200605 and C. coli strain 200606, respectively. WGS confirmed C257T mutation in the gyrA gene and the presence of cmeABC complex conferring resistance to FQs in both strains. Both strains also exhibited tet(O) genes associated with tetracycline resistance. Various virulence genes associated with motility, chemotaxis, and capsule formation were found in both isolates. However, the analysis of virulence genes showed that C. jejuni strain 200605 is more virulent than C. coli strain 200606. The MLST showed that C. jejuni strain 200605 belongs to sequence type ST-5229 while C. coli strain 200606 belongs to ST-5935, and both STs are less common. The phylogenetic analysis clustered C. jejuni strain 200605 along with other strains reported in Korea (CP028933 from chicken and CP014344 from human) while C. coli strain 200606 formed a separate cluster with C. coli (CP007181) from turkey. The WGS confirmed FQ-resistance in both strains and showed potential virulence of both strains. Further studies are recommended to understand the reasons behind the regional distribution (Korea, China, and Vietnam) of such rare STs. Full article

To aid development of phage therapy against Campylobacter, we investigated the distribution of the clustered regularly interspaced short palindromic repeats (CRISPR) systems in fluoroquinolone (FQ)-resistant Campylobacter jejuni. A total of 100 FQ-resistant C. jejuni strains from different sources were analyzed by PCR and DNA sequencing to determine resistance-conferring mutation in the gyrA gene and the presence of various CRISPR systems. All but one isolate harbored 1–5 point mutations in gyrA, and the most common mutation was the Thr86Ile change. Ninety-five isolates were positive with the CRISPR PCR, and spacer sequences were found in 86 of them. Among the 292 spacer sequences identified in this study, 204 shared 93–100% nucleotide homology to Campylobacter phage D10, 44 showed 100% homology to Campylobacter phage CP39, and 3 had 100% homology with Campylobacter phage CJIE4-5. The remaining 41 spacer sequences did not match with any phages in the database. Based on the results, it was inferred that the FQ-resistant C. jejuni isolates analyzed in this study were potentially resistant to Campylobacter phages D10, CP39, and CJIE4-5 as well as some unidentified phages. These phages should be excluded from cocktails of phages that may be utilized to treat FQ-resistant Campylobacter. Full article

Tetrahydroxystilbene glucoside (TSG) is a unique component of the bone-reinforcing herb Radix Polygoni Multiflori Preparata (RPMP). It has the ability to promote bone formation and protect osteoblasts. However, the underlying mechanism remains unclear. To better understand its biological function, we determined TSG’s effect on murine pre-osteoblastic MC3T3-E1 cells by the MTT assay, flow cytometry, FQ-PCR, Western blot, and ELISA. The results showed that TSG caused an elevation of the MC3T3-E1 cell number, the number of cells in the S phase, and the mRNA levels of the runt-related transcription factor-2 (Runx2), osterix (Osx), and collagen type I α1 (Col1a1). In addition, the osteoprotegerin (OPG) mRNA level was up-regulated, while the nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) mRNA levels were down-regulated by TSG. Furthermore, TSG activated the phosphoinosmde-3-kinase/protein kinase B (also known as PI3K/Akt) pathway, and blocking this pathway by the inhibitor LY-294002 could impair TSG’s functions in relation to the MC3T3-E1 cells. In conclusion, TSG could activate the PI3K/Akt pathway and thus promote MC3T3-E1 cell proliferation and differentiation, and influence OPG/RANKL/M-CSF expression. TSG merits further investigation as a potential therapeutic agent for osteoporosis treatment. Full article

The complexity of traditional Chinese medicines (TCMs) is related to their multi-component system. TCM aqueous decoction is a common clinical oral formulation. Between molecules in solution, there exist intermolecular strong interactions to form chemical bonds or weak non-bonding interactions such as hydrogen bonds and Van der Waals forces, which hold molecules together to form “molecular aggregates”. Taking the TCM Puerariae lobatae Radix (Gegen) as an example, we explored four Gegen decoctions of different concentration of 0.019, 0.038, 0.075, and 0.30 g/mL, named G-1, G-2, G-3, and G-4. In order of molecular aggregate size (diameter) the four kinds of solution were ranked G-1 < G-2 < G-3 < G-4 by Flow Cell 200S IPAC image analysis. A rabbit vertebrobasilar artery insufficiency (VBI) model was set up and they were given Gegen decoction (GGD) at a clinical dosage of 0.82 g/kg (achieved by adjusting the gastric perfusion volume depending on the concentration). The HPLC fingerprint of rabbit plasma showed that the chemical component absorption into blood in order of peak area values was G-1 < G-2 > G-3 > G-4. Puerarin and daidzin are the major constituents of Gegen, and the pharmacokinetics of G-1 and G-2 puerarin conformed with the two compartment open model, while for G-3 and G-4, they conformed to a one compartment open model. For all four GGDs the pharmacokinetics of daidzin complied with a one compartment open model. FQ-PCR assays of rabbits’ vertebrobasilar arterial tissue were performed to determine the pharmacodynamic profiles of the four GGDs. GGD markedly lowered the level of AT₁R mRNA, while the AT₂R mRNA level was increased significantly vs. the VBI model, and G-2 was the most effective. In theory the dosage was equal to the blood drug concentration and should be consistent; however, the formation of molecular aggregates affects drug absorption and metabolism, and therefore influences drugs’ effects. Our data provided references for the rational use of Chinese medicines in the clinic, such as the best oral preparation and decoction concentration. Full article

Cervical cancer is the second most common female carcinoma. Current therapies are often unsatisfactory, especially for advanced stage patients. The aim of this study was to explore the effects of tatariside G (TG) on apoptosis in human cervical cancer HeLa cells and the possible mechanism of action involved. An MTT assay was employed to evaluate cell viability. Hoechst 33258 staining and flow cytometry (FCM) assays were used to detect cell apoptosis. The protein expression of phosphorylated JNK, P38, ERK and Akt and cleaved caspase-3 and caspase-9 was evaluated by western blot analysis. Additionally, the mRNA expression of caspase-3 and caspase-9 was measured by fluorescent quantitative reverse transcription-PCR (FQ-RT-PCR). TG notably inhibited cell viability, enhanced the percentage of apoptotic cells, facilitated the phosphorylation of p38 MAPK and JNK proteins and caspase-3 and caspase-9 cracking, downregulated the phosphorylation level of Akt, and increased the loss of MMP and the mRNA expression of caspase-3 and caspase-9. TG-induced apoptosis is associated with activation of the mitochondrial death pathway. TG may be an effective candidate for chemotherapy against cervical cancer. Full article